Determination of the molar absorptivity of NADH.

McComb, Robert B.; Bond, Lawrence W.; Burnett, Robert W.; Keech, Richard C.; Bowers, George N.

doi:10.1093/clinchem/22.2.141

Cited by 131 publications

(48 citation statements)

References 18 publications

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“…NADH, Fld Sq , and Fld Ox were monitored at 340 nm (ε = 6.2 mM −1 cm −1 ), 580 nm (ε = 5.7 mM −1 cm −1 ), and 450 nm (ε = 11.3 mM −1 cm −1 ), respectively. 12,52 The Fld used in the assays as the low-potential electron acceptor was purified in the hydroquinone state as previously described. 5 Fld Hq was exposed to oxygen for a short period of time, upon which the majority (>80%) of the Fld Hq converted to the semiquinone form.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

et al. 2017

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The biological reduction of dinitrogen (N) to ammonia (NH) by nitrogenase is an energetically demanding reaction that requires low-potential electrons and ATP; however, pathways used to deliver the electrons from central metabolism to the reductants of nitrogenase, ferredoxin or flavodoxin, remain unknown for many diazotrophic microbes. The FixABCX protein complex has been proposed to reduce flavodoxin or ferredoxin using NADH as the electron donor in a process known as electron bifurcation. Herein, the FixABCX complex from Azotobacter vinelandii was purified and demonstrated to catalyze an electron bifurcation reaction: oxidation of NADH (E = -320 mV) coupled to reduction of flavodoxin semiquinone (E = -460 mV) and reduction of coenzyme Q (E = 10 mV). Knocking out fix genes rendered Δrnf A. vinelandii cells unable to fix dinitrogen, confirming that the FixABCX system provides another route for delivery of electrons to nitrogenase. Characterization of the purified FixABCX complex revealed the presence of flavin and iron-sulfur cofactors confirmed by native mass spectrometry, electron paramagnetic resonance spectroscopy, and transient absorption spectroscopy. Transient absorption spectroscopy further established the presence of a short-lived flavin semiquinone radical, suggesting that a thermodynamically unstable flavin semiquinone may participate as an intermediate in the transfer of an electron to flavodoxin. A structural model of FixABCX, generated using chemical cross-linking in conjunction with homology modeling, revealed plausible electron transfer pathways to both high- and low-potential acceptors. Overall, this study informs a mechanism for electron bifurcation, offering insight into a unique method for delivery of low-potential electrons required for energy-intensive biochemical conversions.

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Section: ■ Materials and Methodsmentioning

confidence: 99%

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

et al. 2017

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“…The excitation was detected at 340 nm at 25 °C for 3 min by using a spectrophotometer. The mean extinction diminution per min was divided by molar absorptivity of NADH (6317 l mol −1 cm −1 at 340 nm and 25 °C; McComb et al ., ) to determine the concentration of LDH in each sample.…”

Section: Methodsmentioning

confidence: 99%

Acute toxicity and tissue distribution of CdSe/CdS‐MPA quantum dots after repeated intraperitoneal injection to mice

Haque

Seo

et al. 2012

J of Applied Toxicology

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Quantum dots (QDs) are novel tools with multiple biological and medical applications because of their superior photoemission and photostability characteristics. However, leaching of toxic metals from QDs is of great concern. Therefore, for the successful application of QDs in bioscience, it is essential to understand their biological fate and toxicity. We investigated toxicological effects and tissue distribution of mercaptopropionic acid‐conjugated cadmium selenide/cadmium sulfide (CdSe/CdS‐MPA) QDs after repeated intraperitoneal injection into BALB/c mice. The mice were injected every 3 days with various doses of QDs (0, 5, 10 and 25 mg kg−1). The subsequent effects of QDs on plasma levels of various biomarkers were evaluated at different time points (at 0, 1, 4, 7, 10, 13 and 15 days). Various tissue samples (spleen, liver, lung, kidneys, brain, heart and thymus) were collected for toxicity analysis, distribution testing, histopathological examination and inflammation assessment. No abnormal clinical signs or behaviors were recorded but the body weight of mice treated with 25 mg kg−1 QDs was significantly decreased from day 7 compared with control mice. QDs were observed in the liver, spleen, lung and kidneys, but not in brain or heart. Significantly higher levels of lactate dehydrogenase and nicotinamide adenine dinucleotide phosphate oxidase were found in the plasma, liver and spleen. Histopathological examination did not show any tissue toxicity but the levels of interleukin‐6, a pro‐inflammatory marker, were increased in the plasma, liver and spleen. All of these findings provide insight into the observed toxicological effect levels and tissue‐specific distribution of CdSe/CdS‐MPA QDs. Copyright © 2012 John Wiley & Sons, Ltd.

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“…Ligase activities were measured by tracking AMP formation with its conversion to ADP via myokinase and the subsequent oxidation of NADH with the coupling enzymes pyruvate kinase and lactate dehydrogenase (Auburger and Winter, 1992;Schuhle et al, 2003;James et al, 2016). The oxidation of NADH was measured spectrophotometrically at 340 nm (ε = 6220 M −1 cm −1 ) (McComb et al, 1976). The reaction was started by the addition of the acid substrate.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids

James

Kung

Crable

et al. 2019

Environmental Microbiology

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Summary Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane‐1‐carboxylate and catalyses the novel synthesis of benzoate and cyclohexane‐1‐carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane‐1‐carboxylate synthesis. Enzymes involved in the metabolism of cyclohex‐1,5‐diene carboxyl‐CoA to acetyl‐CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane‐1‐carboxylate. Incorporation of 13C‐atoms from 1‐[13C]‐acetate into crotonate, benzoate and cyclohexane‐1‐carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl‐CoA intermediates to formate was detected. Ligases and membrane‐bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl‐CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.

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Determination of the molar absorptivity of NADH.

Cited by 131 publications

References 18 publications

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

The Electron Bifurcating FixABCX Protein Complex from Azotobacter vinelandii: Generation of Low-Potential Reducing Equivalents for Nitrogenase Catalysis

Acute toxicity and tissue distribution of CdSe/CdS‐MPA quantum dots after repeated intraperitoneal injection to mice

Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids

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